The rapid assembly and disassembly of the actin cytoskeleton is central to many cellular functions, including mitosis, cell division, intracellular transport, and the control of cell shape and polarity. Alterations of cytoskeletal proteins are linked to diseases, including cardiovascular and neurodegenerative disorders. The hydrolysis of ATP by actin drives the transition between its monomeric form (G-actin) and filamentous form (F-actin). F-actin is asymmetric, undergoing net association of ATP-actin to the "barbed" end and dissociation of ADP-actin from the "pointed" end. This dynamic process, known as actin filament treadmilling, is regulated in vivo by a plethora of actin-binding proteins (ABPs). ABPs have evolved relatively few actin-binding motifs, including the WASP homology domain-2 (WH2), actin-depolymerizing factor-homology (ADF-H), and calponin-homology (CH) motifs. The long-term goal of this proposal is to understand the structural basis for the interactions of these motifs with G- and F-actin and their role in the regulation of the cytoskeleton. A unifying hypothesis is proposed that holds that ADF-H, WH2, gelsolin, and actin itself, which are generally unrelated, present common structural features that allow them to share a common binding site on actin, consisting of a hydrophobic pocket at the interface between subdomains 1 and 3. In contrast, the CH domain, which is a classical F-actin-binding motif, is not expected to bind in this pocket. Specific aim 1 studies the X-ray structures of complexes of WH2 domains from various disease-related proteins with actin. Specific aim 2 deals with the study of twinfilin, which is composed of two ADF-H domains in tandem. Specific aim 3 focuses on the study of the crystal structure of the CH pair from alpha-actinin, its regulation by PIP2, and its interaction with F-actin. Crystals are available in aims 1 and 3, and micro-crystals are also available in aim 2. The crystallographic and biochemical studies are being complemented by EM studies of twinfilin and a-actinin decorated F-actin, in collaboration with W.J. Lehman at BU. This proposal offers a comprehensive approach to study three of the most important actin-binding motifs in nature. By studying these motifs in parallel we will establish common and distinctive features of their structures and interactions with G- and F-actin that determine their functions in the regulation of the cytoskeleton.